Markus M. Lerch Department of Medicine A

Ernst-Moritz-Arndt Universität Greifswald

Medizinische Klinik der königlichen Universität Greifswald

18591456

550 Jahre Universität Greifswald

Advances in the etiology of chronic pancreatitisEuropean Postgraduate School

in Gastroenterology Prague, April 2010

Chronic Pancreatitis - Etiology

Alcohol Idiopathic Metabolic Anatomical Hereditary Autoimmune

Reasons to discriminate between different etiologies:

Specific treatment options

Inherent co-morbidities

Different cancer surveillance strategies

0

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0 5 10 15 20 25

Alcoholic-CP

Inci

denc

e of

cal

cific

atio

nsP

ropa

bilil

ty in

%

Time after onset of symptoms

Non-Alcoholic CP

Layer et al, Gastroenterology 1994;107:1481- 87

Chronic Pancreatitis – Alcohol Cessation

Chronic Pancreatitis - Alcohol Cessation

0 20 40 60 80 100

Strum et al, 1995

Miyaka et al, 1987

Bornman et al, 1980

Leger et al, 1974

Strum et al, 1971 Alcohol

Stop AlcoholGastard et al, 1973

Prinz et al, 1974

Marks et al, 1980

Hayakawa et al, 1989

Patients with pain [%]

Cu

mu

lativ

e In

cid

en

ce o

f ca

lcifi

catio

ns, %

years after diagnosis of chronic alcoholic pancreatitis

OR 2.0 [CI 1.1-3.8]

Chronic Pancreatitis – Cessation of Smoking Maisoneuve P, Gut 2005; 54: 510-514

Log rank p<0.0001

0 2 4 6 8 10 12 14 16

100

80

60

40

20

0

Smokers

Non-Smokers

Hereditary Pancreatitis

14 year old girl with chronic pancreatitis and R122H-mutation

48 year old women with chronic pancreatitisand R122H-mutation

Hereditary pancreatitis is clinically indistinguishable from other forms and varities of pancreatitis.

1952, first description ofhereditary pancreatitis (autosomal dominant trait).

1996 Discovery of the first mutation associated with hereditary pancreatitis in the cationic trypsinogen gene (PRSS1) by Whitcomb et al.

Hereditary pancreatitis in Germany

MünsterMünster

GreifswaldGreifswald

Weiss F.U. Am J Gastroenterol. 2008;103:2585-8.

Haplotype-Analysis of 10 unrelated families witha R122H mutation from an area of 100 km resulted in 7 different haplotypes. This precludes a Founder-Effect

Block A (17 kb) Block B (62 kb)

Chr. 7: 142,092-142,183 kbPRSS1 PRSS2

D22G

K23R

R116C

K92N

E79K

G83E

R122H/C

V123M

P36R

N29I/T

D100H

L104PC139F

Activation site

Cationic trypsin

0

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50

60

70

80

90

100

0 10 20 30 40 50 60

Cu

mu

lativ

e a

ge a

t ons

et,

%

R122H negativ

N29I A16V

Cu

mu

lativ

e in

cid

enc

e o

f dia

bete

s, %

age, years

0

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30

40

50

60

70

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100

0 20 40 60 80

male female

age, years

Howes N, Lerch MM et al Clinical Gastroenterology and Hepatology 2004; 2: 252-261

Clinical course of Hereditary Pancreatitis

Alcoholic CP

50-70% increased riskfor pancreatic cancer in patients with hereditary pancreatitis. 40% cumulative risk until 70. years of age.

Elimination/Treatmentof causal factors:Smoking, AlcoholHyperlipidemia, Hypercalcemia,Gallstones, Duct stricture,Drugs and Medications age, years

0

10

20

30

40

50

60

0 20 40 60

cumulative pancreatic cancer risk, %

age

at d

iag

nosi

s of

pan

crea

tic c

an

cer 80

70

60

50

40

30

20Non-smokers smokers

p < 0.01

Pancreatic Cancer in Hereditary Pancreatitis

Howes N, Lerch MM et al Clinical Gastroenterology and Hepatology 2004; 2: 252-261;

Lowenfels AB, JAMA 2001; 286: 169-170.

Recurrent (2 or more) episodes of acute Pancreatitis without identifyable cause or etiology or

Idiopathic chronic Pancreatitis - especially in children and young adults under the age of 25 years or

Pancreatitis in a patients with a positive family history of Pancreatitis (one or more first or second degree relatives)

Indications for Genetic Hereditary Pancreatitis testing

I. Ellis, M.M. Lerch, D.C. Whitcomb: Pancreatology 2001:1:405-415

0 5 10 15 20 25 30

0

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60

80

Try

psi

n a

ctiv

ity (

%)

Time (min)

G191R

PRSS2

0 0.5 1 1.5 2

0

20

40

60

80

100

R122C

Wild-Type

pH 8.0, 5 mM Ca2+

Try

psi

n a

ctiv

ity (

%)

Time (h)

Simon et al. J. Biol. Chem. 2002; 277: 5404-5410 Witt et al Nature Genetics 2006; 38: 668-73.

Wild-Type

PRSS1

Pathophysiological role of two mutant trypsinogens:Decreased autoactivation

wt R122C

reducing non-reducing

60

42

30

22

17

kDa

wt R122C

Simon P. et al. J. Biol. Chem. 2002; 277: 5404-5410

SHI

CH2

ICIH

=COO-H3N+-

PRSS1

Cystein

Trypsinogen

Wild type G191R

0 2 5 10 0 2 5 10 min

Trypsin

insertion of a new cleavage site EGGKD / ERGKD

PRSS2

Witt et al Nature Genetics 2006; 38: 668-73.

The phenotype of two novel trypsinogen mutations

II:1 II:4II:262y

II:354y

III:612y

III:517y

III:424y

III:325y

III:230y

III:131y

II:552y

II:6 II:745y

I:187y

I:169y diab.

Symbole

o. A.

Pancreatitis

test for R122C

carrier

PRSS1

Simon P. et al. J. Biol. Chem. 2002; 277: 5404-5410

Screening of Exon 4

for G191R mutations:

ICP/HP 17/1414 (1.2%)

ACP 4/405 (1.0%)

CONTROL 157/4581 (3.5%)

PRSS2

Witt et al Nature Genetics 2006; 38: 668-73.

0 0.5 1 1.5 2

0

20

40

60

80

100

Wild type

pH 8.0, 5 mM Ca2+

Try

ps

in A

ctiv

ity

(%)

time (h)

Kereszturi et al. Hum Mutation 2009; 30: 575-82

R116C Trypsin – Retention and ER Stress

R116C

I

II

III

IV

63y 62y

48y 44y 40y 38y 36y

17y 15y

35

25

Homogenate Pellet SupernatantkDa

wtwt wtwt wtwt R116CR116CR116CR116CR116CR116C

Expression in 293T cells

100

70BiP (78 kDa)

wt R116A R116C

PRSS-Mutations: „gain or loss of function“ ?

Autoactivation Autolysis Cathepsin B-ind. activation

A16V ? ? ?

D19A ▲

D22G ▲

K23R ▲

N29I ▲ ◄► ◄►

E79K ▼ ◄►

R122C ▼ ▼ ▼

R122H ▲ ▼ ◄►

XXX ◄► ◄► ◄►

R116C ◄► ◄► ◄►

G191R ▼ ▲ ?

▼ intracellular processing

▼ intracellular processing

Sporadic point mutations in the PRSS1- Gen in idiopathic chronic Pancreatitis

In 5 of 50 Patients with idiopathic Pancreatitis (10%) mutations inthe cationic Trypsinogen gene were found.

10%

90%

35%

65%

Affected Patients represented 35%of all patients under 25 years. Simon P, Weiss F.U. et al JAMA. 2002;288:2122

Sporadic point mutations in the PRSS1- The French CohortRebours V Am J Gastroenterol 2008; 103: 111-119, Rebours AJG 2009; 104: 2312Masson E et al Clin Gastroenterol Hepatol 2008; 6: 82-88

In 78 families with hereditary pancreatitis and 200 individuals (6673 patients years) PRSS1 mutations were detected in 68%. R122H: 78%, N29I: 12% and others 10%.

Cumulative risk of pancreatic cancer was 11% at the age of 50 and 49% at the age of 75.

Smoking and diabetes mellitus are the main risk factors.

Trypsinogen copy number variations are present in 6% of idiopathic chronic pancreatitis cases but are unrelated to familial chronic or tropical calcifying pancreatitis.

Chymotrypsin C (CTRC) variants that diminish activity or secretion are associated with chronic pancreatitis Rosendahl et al Nature Genetics 2008; 40: 78-82

Chymotrypsin C is a trypsin-degrading enzyme. Alterations in the CTRC Gene at position p.R254W and p.K247_R254del are present in 30 out of 901 (3.3%) pancreatitis individuals but only in 21 of 2804 controls (0.7%).

Functional analysis showed impaired activity or reduced secretion indicating that loss of function alterations in CTRC predispose to pancreatitis.

Pancreatic secretory Trypsin Inhibitor (PSTI, SPINK-1)

Model by A. Brunskil & W. F. Furey Model by A. Brunskil & W. F. Furey

Witt et al, (Nat. Genet., 2000) Mutations in 23% of children with idiopathic chronic Pancreatitis autosomal-recessive disorder

Pfützer et al, (Gastroenterology, 2000)Mutations in idiopathic chronic Pancreatitis (25%), hereditary Pancreatitis and inthe healthy population (2%). Modifier - Gene, risk of pancreatitis < 1%

Bhatia et al, Schneider et al, (Gastroenterology, 2002)Mutations in tropical calcifying pancreatitis (up to 44%) and in ‘Fibrocalculous Pancreatic Diabetes mellitus‘ (55%). Risk factor for tropical Pancreatitis and Diabetes mellitus

SPINK1-Mutations in Patients with Hereditary PancreatitisSymbols

Empty symbol

chronic Pancreatitis

positiv for PRSS1 Mutation

positiv for N34S Mutation

Asymptomatic carrier

wt

IV:1 8

IV:25

I:154

I:2 81

II:164

II:3 61

II:5 60

III:2 36

II:2 II:4 69

II:7 50

III:516

II:6 68

III:613

II:8 46

III:3 35

III:1 III:4

I:1 I:287

II:259

II:359

II:462

III:432

II:150

III:128

III:226

III:330

I:245

II:1

I:145

SPINK1 Mutations (N34S) are found among Pancreatitis patients as well as among healthy carriers of Trypsinogen mutations. Weiss F.U. et al. J. Med. Gen. 2003;40:e40,1-5

I.

II.

III.

SPINK1 Mutations in Hereditary PancreatitisP

hen

oty

pe,

% o

f to

tal

Cumulative Incidence of Pancreatitis

Age at disease onset, years0 10 20 30 40 50 60

0

20

40

60

80

100

PRSS1, SPINK wt

PRSS1, SPINK N34S

0 10 20 30 40 50 60

20

40

60

80

100

mild Pancreatitis

severe Pancreatitis

0 10 20 30 40 50 600

20

40

60

80

100

No Diabetes

Diabetes

SPINK1 Mutations have no influence on the severity of clinical disease courseof hereditary pancreatitis Weiss F.U. et al. J. Med. Gen. 2003;40:e40,1-5

Hyderabad Calicut Total

Patients/Controls Patients/Controls Patients/Controls(n=140) (n=155) (n=166) (n=175) (n=306) (n=330)

Leu26Val 0.48 0.30 0.45 0.28 0.46* 0.29* * p= 0.013

Ser53Gly 0.10 0.06 0.09 0.04 0.09* 0.04** p= 0.152

Association of CTSB Polymorphisms with Tropical Calcific Pancreatitis

Mahurkar et al. Gut. 2006 55:1270-5.

ethnic origin: Dravidian

N34S positive N34S negative Controls(n=134) (n=172) (n=330)

Leu26Val 0.45 0.46 0.29

His110His111

E64d

n C/C C/G G/G pGpG

(Mahurkar)

Patients 64 23 31 10 0,398 (51/128) 0,46

Controls 100 30 44 26 0,480 (96/200) 0,29

X2 p-Value 0,428 0,578 0,117 0,147 0,013

OR 0,764 0,836 1,897 0,718 2,09

95% CI0,372 -

1,5690,424 -

1,6480,792 -

4,621 0,45 - 1,15 1,55 - 2,81

CTSB Val26 mutation in German ICP Patients

Weiss et al. Gut. 2007 56(9):1322-3.

Population Ethnic origin (n) pG (Val26)

CEPH Mixed Caucasian 92 0,320

Pooled_CEPH Caucasian 94 0,323

HapMap-CEU European 55 0,355

SC_95_C Caucasian 45 0,367

HapMap-YRISub-Saharan African 52 0,394

TSC_42_C Caucasian 41 0,430

JBIC-allele Japanese 732 0,493

HapMap-JPT Asian 44 0,523

HapMap-HCB Asian 43 0,547

1198 0,452 ± 0,042

Cathepsin B mutation Leu26Val in pancreatitis

see refSNP ID: rs12338 at http://www.ncbi.nlm.nih.gov /SNP/snp_ref.cgi?rs=12338

Mahurkar et al. Gut 2006 55:1270-5

N34S positive N34S negative Controls(n=134) (n=172) (n=330)

Leu26Val 0.45 0.46 0.29

Weiss et al. Gut 2007 56:1322-3

Chronic Pancreatitis and CFTR Mutations

F 508 Mutation

Kerem et al. Science

1989; 245: 1073-80

Kerem et al. Science

1989; 245: 1073-80

One third of patients (n=27) with idiopathic pancreatitis carry CFTR-Mutations (Risk x 80). J. Cohn et al. New Engl J Med 1998;339:653-58

CFTR Mutations represent a risk factor for chronic pancreatitis in patients without a history of alcohol abuse (19% of n = 60), but not for those with alcoholic pancreatitis (8.5% of n = 72).N. Sharer et al. New Engl J Med 1998;339:645-52

Weiss FU Gut 2005; 54: 1456-1460

CFTR allele frequency

12/66 Patients with CFTR Mutations, 8/66 Patients with T5 Allels

Prevalence of gene mutations in chronic pancreatitis

Idiopathic pancreatitis

45.5%

Trypsinogen mutations

10%

SPINK-1 mutations

15.2%

T5 Allels

12.1%

CFTR mutations

18.2%

Chymotrypsin C mutations

10%

Metabolic Chronic Pancreatitis – Causal treatment

Hyperparathyroidism: Hyperthyreoidims leads to increased serum calcium levels, what is associated with an increased risk of pancreatitis.

The incidence of chronic pancreatitis is between 1.5 - 7%.

Early parathyreoidectomy leads to resolution of symptoms.

Bess MA JAMA 1980; 243: 246-247; Russel CF Br. J. Surg. 1982; 69: 244-247;

Hyperlipidemia (apoCII deficiency, lipoprotein lipase deficiency)

Serum triglyercide levels > 1000 mg/dlIncidence extremely lowTreatment which maintains TG below 500 mg/dl leads to resolution of symptoms.

Toskes PP, Gastroenterol Clin North Am 1990; 19: 783-791.

Summary and ConclusionSummary and Conclusion

Hereditary chronic and idiopathic chronic pancreatitis are associated with mutations in the trypsinogen gene, the SPINK-1 Gene, the chymotrypsin C gene and the CFTR gene.

More genes will surely be identified.

The pathophysiological impact of these gene mutations are not

completety understood and further experiments are warranted.

Etiologies of pancreatitis already treatable (such as autoimmune pancreatitis etc. ) need to be distinguished from pancreatitis varieties that are not yet treatable – but may become so.

In hereditary pancreatitis (trypsin mutations) smoking cessation may reduce the risk of pancreatic cancer. Other surveillance strategies are, however, urgently needed.

Julia MayerleUli WeissPeter SimonAli AghdassiGabriele Sauter

www.pancreas.de

Julia MayerleUli WeissPeter SimonAli AghdassiGabriele Sauter

www.pancreas.de

Chronic Pancreatitis - Alcohol Cessation

0

20

40

60

80

100

Bicarbonate Lipase Chymotrypsin

Stop Alcohol

Alcohol

Gullo et al, Gastroenterology 1988;95:1063-68

Out

put i

n %

of

initi

al

n= 18 vs. 14 pt∆ 4-11 yearsp<0.01

P. Simon, F.U. Weiss et al. J Biol Chem. 2002;277:5404-10

Diagnosis and Screening for Hereditary Pancreatitis

A C G C C N G C G T G T

C T

Sequence in Exon 3 ArgRSS1 wt AAC-GCC-CGC-GTG-T CysR-122-C AAC-GCC-TGC-GTG-T

Afl III BstU I

Wild

type

Wild

type

R122H

R122H

R122C

R122C

Contro

l

Contro

l

Today, restriction enzyme digest with BstU I represents the most extensive initial screening test for hereditary pancreatitis.

Natural Course of Alcoholic Chronic Pancreatitis -

Modified according to R Ammann, Schweiz Rundsch Med Prax. 1970; 59: 792-5.

Pain

Serum Enzyme Elevation

Exocrine Pancreatic Function

Stage 1 (early) Stage 2 Stage 3

5 10 15 years